A method and application for visual and rapid detection of the content of azodicarbonamide in food additives

Abstract

A visual rapid detection method for the content of azodicarbonamide as a food additive, comprising the following steps: (1) Weigh a certain amount of food, dissolve it successively with dimethyl sulfoxide and distilled water, adsorb it with activated carbon, filter it under reduced pressure, take the filtrate, and then filter it with an organic phase microporous filter membrane to obtain a test solution; (2) For the visual rapid detection method to determine the content of azodicarbonamide in the test solution, one of the key steps is to use a smartphone application - a color identifier to extract its RGB value. This method is simple to operate, has good repeatability, and low cost, meeting the detection requirements for the content of azodicarbonamide as an additive in the food industry.

Description

Technical Field

[0001] The present invention belongs to the field of food additive detection, and particularly relates to a visual rapid detection method for the content of azodicarbonamide in food. Background Art

[0002] In order to improve the color, aroma, taste and quality of food, preserve food, and prevent spoilage, so as to improve the economic benefits of food production, merchants often add food additives in the food production process.

[0003] Wheat flour is one of the most important food raw materials, famous for the diversity, color and taste of its food. In many countries, products related to wheat flour account for a large proportion in the household diet structure. Now, in order to meet the actual needs, the whiteness and gluten of wheat flour are often improved by adding food additives.

[0004] As early as 1940, azodicarbonamide (ADA) was widely used as a bleaching agent and foaming agent in the production fields of rubber products and foam plastics. Now, due to its oxidation and bleaching properties, ADA has been widely used as a bleaching agent, gluten fortifier or dough conditioner in the wheat flour industry in many countries. This is because ADA can oxidize the sulfhydryl groups (—SH) in wheat flour to form disulfide bonds (S—S), enabling the protein gold chains to be interconnected to form a network, thereby improving the dough forming ability and enhancing the flexibility and toughness of baked foods. In addition, ADA can also oxidize the natural pigments in wheat flour to increase the whiteness of wheat flour.

[0005] Azodicarbonamide (ADA) itself has low acute toxicity and does not directly react with wheat flour, but it can react with wet wheat flour as an oxidant to be converted into biurea (BIU) and semicarbazide (SEM). SEM is a metabolite of the veterinary drug furacilin and has strong carcinogenic, teratogenic and mutagenic effects. It is reported that under high-temperature environments, such as baking, SEM has genotoxicity and carcinogenicity in vitro. In addition, the World Health Organization has also reported that exposure to ADA in the workplace may cause respiratory problems, allergies and asthma. Therefore, long-term consumption of related products containing excessive ADA will cause serious harm to the human body. Currently, the addition of ADA in food is prohibited in the European Union and regions and countries such as Australia, New Zealand, Singapore and Japan, while in the United States, Canada and China, the maximum allowable level of ADA in wheat flour is 45 mg / kg (ppm).

[0006] So far, many analytical techniques have been developed for the detection of azodicarbonamide (ADA) in wheat flour, mainly including direct detection techniques and indirect detection techniques. The target analyte of direct detection techniques is ADA, while the target analytes of indirect detection techniques are the decomposition products of ADA, namely biurea (BIU) and semicarbazide (SEM). Direct detection techniques include surface-enhanced Raman spectroscopy, infrared spectroscopy, high-performance liquid chromatography, capillary electrophoresis, and colorimetric analysis. Indirect detection techniques include enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry, which can determine semicarbazide converted from ADA.

[0007] With the frequent occurrence of food safety incidents, rapid detection techniques have developed rapidly. Existing techniques for detecting ADA in wheat flour have various drawbacks, such as complex pretreatment, time-consuming, high equipment costs, long detection times, and difficulty in popularization at the county and district-level regulatory units, which may limit their practical applications in determining ADA. At the same time, all along, in order to control the effectiveness of legal regulations and ensure the edible safety of wheat flour, there is still a need to develop a relatively rapid and visual on-site detection method for ADA in wheat flour samples.

[0008] In this invention, indigo disulfonate sodium is used as a reducing agent and a chromogenic agent, and ammonium metavanadate is used as a catalyst. When ADA with an oxidizing effect is added, after the catalytic action of ammonium metavanadate and heating at 45 °C for 35 min, indigo disulfonate sodium changes from its original blue color to colorless. In this method, there is a good linear relationship between the ADA concentration in the range of 0 - 40 μg / mL and the absorbance value A (at 610 nm). Then, by taking a photo with a mobile phone and using a mobile phone software - color identifier to extract the RGB values in the solution, a linear relationship is established between the R / B ratio and the ADA concentration in the range of 0 - 40 μg / mL. And the practical application value of this method is proved through interference experiments and standard addition recovery experiments. Summary of the Invention

[0009] Based on the above background technology, the present invention provides a visual detection method for the content of azodicarbonamide in food, which is characterized by including the following steps:

[0010] (1) Using indigo disulfonate sodium solution as a reducing agent and ammonium metavanadate solution as a catalyst, and using a smartphone application - color identifier to extract the RGB values of azodicarbonamide solutions with different concentrations, establishing a linear relationship between azodicarbonamide and the R / B ratio, and establishing the linear equation of the standard working curve.

[0011] (2) Weigh 0.8000 g of the food sample, add the solvent, shake to dissolve, centrifuge and separate, take the supernatant, adsorb with activated carbon, filter under reduced pressure, take the filtrate, and then filter with an organic phase microporous membrane to obtain the test solution. As described in step (1), using indigo disulfonate solution as the reducing agent and ammonium metavanadate solution as the catalyst, use the smartphone application - color identifier to extract its RGB value. According to the standard working curve linear equation in step (1), the content of azodicarbonamide in the test solution can be obtained.

[0012] 2. In step (1): The volume of the added indigo disulfonate solution as the reducing agent is 1.1 mL and its concentration is 0.001 mol / L, and the volume of the added ammonium metavanadate solution as the catalyst is 1.0 mL and its concentration is 150 μg / mL.

[0013] 3. In step (1): There is a good linear relationship between the concentration (x) of the azodicarbonamide solution in the range of 0 - 40 μg / mL and the R / B ratio (y), and the standard working curve linear equation is y = 0.0144x + 0.6104.

[0014] 4. In step (2): The solvent used to dissolve the food sample is 5 mL of dimethyl sulfoxide first, and then 15 mL of distilled water.

[0015] 5. In step (2): The specification of the organic phase microporous membrane is 0.45 μm.

[0016] The visual detection method for the content of azodicarbonamide in the above food has the following characteristics compared with the prior art: Using indigo disulfonate as the reducing agent and color developer, and ammonium metavanadate as the catalyst. When adding ADA with an oxidizing effect, after the catalytic action of ammonium metavanadate and heating at 45 °C for 35 min, indigo disulfonate changes from the original blue to colorless. In this method, there is a good linear relationship between the ADA concentration in the range of 0 - 40 μg / mL and the absorbance value A (at 610 nm). Then, by taking a photo with the mobile phone and using the mobile phone software - color identifier to extract the RGB value in the solution, a linear relationship between R / B and the ADA concentration in the range of 0 - 40 μg / mL is established, and the practical application value of this method is proved through interference experiments and standard addition recovery experiments. Description of the Drawings

[0017] Figure 1 Standard curve of azodicarbonamide (ADA) concentration vs. R / B value.

[0018] Figure 2 Result of non - detection of azodicarbonamide content in flour sample by high - performance liquid chromatography. Detailed Embodiments

[0019] The following examples are for further illustration of the present invention rather than limitations thereof.

[0020] Example 1.

[0021] (1) Establishment of the linear equation of the standard working curve

[0022] In a colorimetric tube, use a pipette to successively add 1.0 mL of ammonium metavanadate standard solution with a concentration of 150 μg / mL, 1.1 mL of indigo disulfonate solution with a concentration of 0.001 mol / L, and 4.5 mL of azodicarbonamide ADA solutions with each concentration gradient (0, 1, 5, 10, 20, 30, 40 μg / mL), 3 mL of 85% phosphoric acid solution, shake well and start timing. After reacting for 35 min under the condition of heating in a 45°C constant temperature water bath, load the ADA solutions with each concentration gradient into a colorimetric cell, take pictures with a mobile phone, and extract the RGB color values of the solution under the smartphone application - Color Recognizer to establish the linear relationship between the concentration of each ADA and the R / B ratio (see attachment Figure 1 ), the linear regression equation is y = 0.0144x + 0.6104, and the correlation coefficient is r 2 = 0.9912. Based on the 3σ / k rule, the detection limit is 4.518 μg / mL.

[0023] (2) Pretreatment of flour samples

[0024] Weigh 0.8000 g (accurate to 0.1 mg) of wheat flour into a 250 mL conical flask, add 5 mL of dimethyl sulfoxide solution to dissolve it, then add 15 mL of distilled water, seal it and place it on a shaker to shake at a speed of 200 r / min at room temperature for 15 min, let it stand for 5 min, then transfer the solution into a 10 mL centrifuge tube, centrifuge at a speed of 4000 rpm for 10 min, take the supernatant, add 0.2000 g of activated carbon, stir evenly and let it stand for adsorption for 20 min, then filter it with a Buchner funnel, take the filtrate, and filter it again with a 0.45 μm organic phase microporous filter membrane to obtain the blank matrix extraction solution for standby.

[0025] (3) Analysis of flour samples and spiking recovery experiments

[0026] Using the visual rapid detection method established by the present invention, determine the content of azodicarbonamide in commercially available wheat flour samples. It is observed that the color of the solution is not much different from that of the blank solution distilled water (attachment Figure 2 ), which indicates that no ADA is detected in the commercially available wheat flour samples. Use a high-performance liquid chromatograph to detect ADA in commercially available wheat flour samples according to the national standard GB-5009.283-2021, and the results are also all undetected. This proves the accuracy of the detection of this method from the side.

[0027] Using the extract of the flour sample, three groups of spiked recovery experiments were conducted in parallel. According to the ADA concentration - R / B ratio, the average recovery rate was calculated to be 98.98% - 102.38% (see Table 1).

[0028] Table 1 Results of spiked recovery experiments

[0029]

[0030] Compared with the results of high - performance liquid chromatography and the results of spiked recovery experiments, it shows that the method for visual and rapid detection of the content of azodicarbonamide in food additives established by the present invention has high accuracy and good precision, and can be used for on - site visual and rapid detection of ADA in wheat flour.

Claims

1. A rapid detection method for the content of azodicarbonamide as an additive in flour, characterized in that, it comprises the following steps: (1) Establishment of the linear equation of the standard working curve: In 7 colorimetric tubes, pipette 1.0 mL of ammonium metavanadate standard solution with a concentration of 150 μg / mL, 1.1 mL of indigo disulfonate sodium solution with a concentration of 0.001 mol / L, and 3 mL of 85% phosphoric acid solution in sequence. Then add 4.5 mL of azodicarbonamide (ADA) solutions with concentration gradients of 0, 1, 5, 10, 20, 30, and 40 μg / mL. Shake the solutions in the 7 colorimetric tubes, and place them in a constant temperature water bath at 45 °C for reaction for 35 min. Then transfer the solutions into 7 colorimetric cuvettes, take pictures with a mobile phone, and extract the RGB color values of the solutions under the smartphone application - Color Recognizer. Establish the linear relationship between the concentration of ADA and the R / B ratio. The linear regression equation of the standard working curve is y = 0.0144x + 0.6104; (2) Determination of the concentration of azodicarbonamide (ADA) in the sample: Weigh 0.8000 g of flour sample, first dissolve it with 5 mL of dimethyl sulfoxide, then add 15 mL of distilled water, shake to dissolve, centrifuge and separate, take the supernatant, adsorb it with activated carbon, filter it under reduced pressure, take the filtrate, and then filter it through a 0.45 μm organic phase microporous filter membrane to obtain the test solution. As described in step (1), the added volume of ammonium metavanadate solution is 1.0 mL with a concentration of 150 μg / mL, the added volume of indigo disulfonate sodium solution is 1.1 mL with a concentration of 0.001 mol / L, and 3 mL of 85% phosphoric acid solution. Use the smartphone application - Color Recognizer to extract its RGB value. According to the linear equation of the standard working curve established in step (1), the content of azodicarbonamide in the test solution can be obtained.

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